Chemical Constituents and Their Biological Activities from Genus Styrax

Plants from the genus Styrax have been extensively used in folk medicines to treat diseases such as skin diseases and peptic ulcers and as an antiseptic and analgesic. Most Styrax species, especially Styrax tonkinensis, which is used as an expectorant, antiseptic, and analgesic in Chinese traditional medicine, could screen resin after external injury. Styrax is also used in folk medicines in Korea to treat sore throat, bronchitis, cough, expectoration, paralysis, laryngitis, and inflammation. Different parts of various Styrax species can be widely employed for ethnopharmacological applications. Moreover, for ethnopharmacological use, these parts of Styrax species can be applied in combination with other folk medicines. Styrax species consist of versatile natural compounds, with some of them exhibiting particularly excellent pharmacological activities, such as cytotoxic, acetylcholinesterase inhibitory, antioxidant, and antifungal activities. Altogether, these exciting results indicate that a comprehensive review of plants belonging to this genus is essential for helping researchers to continuously conduct an in-depth investigation. In this review, the traditional uses, phytochemistry, corresponding pharmacological activities, and structure–activity relationships of different Styrax species are clarified and critically discussed. More insights into potential opportunities for future research are carefully assessed.


Introduction
The genus Styrax has a widespread but dispersive distribution. It is found in East Asian, American, and Mediterranean regions. It is the largest genus of the Styracaceae family and contains approximately 130 species [1]. Styrax stands out from other genera in this family because it produces a resinous material known as benzoin resin. This resin is typically released when the bark is injured by sharp objects. It has been utilized in various regions across the globe for its aromatic properties, being commonly used in perfumes and cosmetics, and Styrax species have traditionally been used in herbal medicines for the treatment of various diseases [2]. Of note, many Styrax species, especially S. tonkinensis, which is used as an expectorant, antiseptic, and analgesic in Chinese traditional medicine, could screen resin after exterior injury [3,4]. As a folk medicine in Korea, S. japonica is used to treat cough, bronchitis, sore throat, inflammation, paralysis, laryngitis, and expectoration [5][6][7]. The resin from Styrax, mixed with other antibiotic substances and hardening material, is also indicated in Islamic medicine as working as a good dental restorative material [2]. The flower of S. japonicus sieb. et Zucc. is used in Chinese folk medicine to relieve pain such as sore throat pain and toothache [8]. The leaves and roots of Styrax suberifolium are typically used as traditional medicines in China to cure rheumatic diseases [9].
Furthermore, the extensive investigation of pharmacologically active compounds derived from various Styrax species has been ongoing for several decades. While numerous Styrax species have been studied, S. obassia and S. japonica have emerged as the most extensively researched species, encompassing studies ranging from phytochemistry to comprehensive pharmacological investigations ( Figure 1). An example of the pharmacological potential of Styrax species was the inhibitory effect of benzofurans extracted from Styrax agrestis A. Chev. on acetylcholinesterase (AChE) in vitro [10]. Triterpenoids isolated from the resin of S. tonkinensis (Pier.) Craib showed promising antiproliferative and differentiation effects on human leukemia HL-60 cells [4]. Additionally, the hydroalcoholic extracts of S. camporum Pohl demonstrated effectiveness in reducing chromosome and DNA damage [11]. Another notable finding was the promotion of estrogen biosynthesis by egonol gentiobioside and egonol gentiotrioside from Styrax perkinsiae through the action of aromatase [12].
suberifolium are typically used as traditional medicines in China to cure rheumatic diseases [9]. Furthermore, the extensive investigation of pharmacologically active compounds derived from various Styrax species has been ongoing for several decades. While numerous Styrax species have been studied, S. obassia and S. japonica have emerged as the most extensively researched species, encompassing studies ranging from phytochemistry to comprehensive pharmacological investigations ( Figure 1). An example of the pharmacological potential of Styrax species was the inhibitory effect of benzofurans extracted from Styrax agrestis A. Chev. on acetylcholinesterase (AChE) in vitro [10]. Triterpenoids isolated from the resin of S. tonkinensis (Pier.) Craib showed promising antiproliferative and differentiation effects on human leukemia HL-60 cells [4]. Additionally, the hydroalcoholic extracts of S. camporum Pohl demonstrated effectiveness in reducing chromosome and DNA damage [11]. Another notable finding was the promotion of estrogen biosynthesis by egonol gentiobioside and egonol gentiotrioside from Styrax perkinsiae through the action of aromatase [12].
Despite significant progress in discovering natural compounds from Styrax species and elucidating their potential pharmacological activities, there is still a need for a comprehensive and focused discussion of this rapidly growing research area. With our continuous interests in natural products discovery and pharmacological research [13][14][15][16], our aim is to provide researchers with a convenient and comprehensive resource that offers detailed and concise profiles of the Styrax genus. This review encompasses the examination of structural diversity and the pharmacological and biological significance and presents the exciting future research prospects in this field.  Despite significant progress in discovering natural compounds from Styrax species and elucidating their potential pharmacological activities, there is still a need for a comprehensive and focused discussion of this rapidly growing research area. With our continuous interests in natural products discovery and pharmacological research [13][14][15][16], our aim is to provide researchers with a convenient and comprehensive resource that offers detailed and concise profiles of the Styrax genus. This review encompasses the examination of structural diversity and the pharmacological and biological significance and presents the exciting future research prospects in this field.
To exploit the antibacterial activity of the aerial parts of S. pohlii, different fractions, especially those extracted using n-hexane, EtOAc, n-BuOH, and methanol, were evaluated against Haemophilus influenzae, Pseudomonas aeruginosa, S. pyogenes, Streptococcus pneumoniae, and Klebsiella pneumoniae. The broth microdilution method was used for measuring the minimum inhibitory concentration (MIC). Among the fractions, the n-hexane fraction exhibited excellent antibacterial activity against Gram-positive S. pneumoniae (MIC = 200 µg/mL). The MIC values of compounds 4 and 17 (400.0 µg/mL) against P. aeruginosa and S. pneumoniae were the best [61]. especially those extracted using n-hexane, EtOAc, n-BuOH, and methanol, were evaluated against Haemophilus influenzae, Pseudomonas aeruginosa, S. pyogenes, Streptococcus pneumoniae, and Klebsiella pneumoniae. The broth microdilution method was used for measuring the minimum inhibitory concentration (MIC). Among the fractions, the n-hexane fraction exhibited excellent antibacterial activity against Gram-positive S. pneumoniae (MIC = 200 µg/mL). The MIC values of compounds 4 and 17 (400.0 µg/mL) against P. aeruginosa and S. pneumoniae were the best [61]. By conducting the radial growth-inhibition experiment, the antifungal activities of compounds from the bark of S. suberifolius against three plants' fungal pathogen, namely, Phomopsis cytospore, Fusarium oxysporum, and Alternaria Solani, was exhibited. Compounds 144, 145, and 146 exhibited selective suppressive activities against the tested fungi. Notably, compound 146 was a significantly effective inhibitor of Phomopsis cytospore at 100.0 µg/mL, with an inhibition rate of 86.72% [40].

Induction of Apoptosis
In 2002, a nonradioactive assay was established for measuring aromatase activity by using human ovarian granulosa KGN cells. Lignans 6 and 7 exhibited approximately 1.62-and 1.95-fold increases, respectively, in 17 β-estradiol biosynthesis at 10 µM, and significantly improved 17 β-estradiol biosynthesis by approximately 1.53-and 1.71-fold, respectively, in 3T3-L1 preadipocyte cells ( Figure 7). Moreover, egonol gentiotrioside increased serum estrogen levels in ovariectomized rats. These results suggested that these two lignans induce estrogen biosynthesis through the allosteric regulation of aromatase activity [12]. In 2002, a nonradioactive assay was established for measuring aromatase activity by using human ovarian granulosa KGN cells. Lignans 6 and 7 exhibited approximately 1.62and 1.95-fold increases, respectively, in 17 β-estradiol biosynthesis at 10 µM, and significantly improved 17 β-estradiol biosynthesis by approximately 1.53-and 1.71-fold, respectively, in 3T3-L1 preadipocyte cells ( Figure 7). Moreover, egonol gentiotrioside increased serum estrogen levels in ovariectomized rats. These results suggested that these two lignans induce estrogen biosynthesis through the allosteric regulation of aromatase activity [12]. In 2011, Liu et al. screened their library of plant extracts through a high-throughput assay. They found that the EtOAc extract of S. agrestis fruits exhibited significant inhibitory activity against AChE. They proved that two active subfractions were responsible for this inhibition and further isolated 13 compounds from the EtOAc extract. Later, they ex-  In 2011, Liu et al. screened their library of plant extracts through a high-throughput assay. They found that the EtOAc extract of S. agrestis fruits exhibited significant inhibitory activity against AChE. They proved that two active subfractions were responsible for this inhibition and further isolated 13 compounds from the EtOAc extract. Later, they examined the selectivity and inhibitory potency of benzofurans on hAChE, BChE, and EeAChE by using the improved Ellman's colorimetric method ( Figure 8). Some egonol derivatives were synthesized through chemical modifications to clearly understand the structureactivity relationships. According to the results, the inhibition ratio affects the bulkiness and length of the alkyl ester group. In particular, compounds 50-53 exhibited inhibitory activity against AChE (IC 50 = 1.4-3.1 µM). Compound 50 at 100.0 µM displayed obvious inhibition of Aβ aggregation (77.6%). Liu et al.'s SAR (Structure-Activity Relationships) studies indicated that compounds exhibiting anti-AChE activity are observed with the incorporation of alkyl chains consisting of more than three carbon units, the furan ring, and the ester group. Molecular docking studies proposed a binding site for this class of compound on AChE and identified multiple key residues at the peripheral site that are crucial for mediating the inhibitory effect [10]. The anti-AChE and antifungal activities of two novel polyketides, 156 and 157, were also tested through TLC bioautographic assays. The results indicated that compound 156 could inhibit AChE activity [57].

Acetylcholinesterase Inhibitors and Structure-Activity Relationships
In 2011, Liu et al. screened their library of plant extracts through a high-throughput assay. They found that the EtOAc extract of S. agrestis fruits exhibited significant inhibitory activity against AChE. They proved that two active subfractions were responsible for this inhibition and further isolated 13 compounds from the EtOAc extract. Later, they examined the selectivity and inhibitory potency of benzofurans on hAChE, BChE, and EeAChE by using the improved Ellman's colorimetric method ( Figure 8). Some egonol derivatives were synthesized through chemical modifications to clearly understand the structure-activity relationships. According to the results, the inhibition ratio affects the bulkiness and length of the alkyl ester group. In particular, compounds 50-53 exhibited inhibitory activity against AChE (IC50 = 1.4-3.1 µM). Compound 50 at 100.0 µM displayed obvious inhibition of Aβ aggregation (77.6%). Liu et al.'s SAR (Structure-Activity Relationships) studies indicated that compounds exhibiting anti-AChE activity are observed with the incorporation of alkyl chains consisting of more than three carbon units, the furan ring, and the ester group. Molecular docking studies proposed a binding site for this class of compound on AChE and identified multiple key residues at the peripheral site that are crucial for mediating the inhibitory effect [10]. The anti-AChE and antifungal activities of two novel polyketides, 156 and 157, were also tested through TLC bioautographic assays. The results indicated that compound 156 could inhibit AChE activity [57].
With further investigation of the SJSZ glycoprotein (38 kDa), Lee and Kim proved that this glycoprotein modulates IFN-γ, IL-2, and IL-12 expression in cyclophosphamide (CTX)-induced Balb/c mice. The glycoprotein counteracted the CTX-induced immunosuppressive effects. It effectively restored the spleen and thymus weights to normal levels and enhanced the phagocytic activity of peritoneal macrophages in response to CTX. Furthermore, the SJSZ glycoprotein exerted regulatory effects on the proliferation of T and B lymphocytes, cytotoxicity of NK cells, and production of key cytokines (IIFN-γ, L-2, and IL-12). Additionally, it improved the activity of antioxidant enzymes (e.g., SOD, CAT, and GPx) [64].

Matrix Metalloproteinase's Activity
Some triterpenoids (95-97, 100) were isolated and further tested the Matrix Metalloproteinases (MMPs)' activity of the methylene chloride soluble fraction of a methanol extract from the stems of S. japonica. Among them, 95 and 100 displayed effective cytotoxic activities against human dermal fibroblasts (IC 50 = 20.0 and 1.12 µM, respectively) ( Figure 9).
In addition, 96 and 97 exhibited no cytotoxicity for the same cells at the test dose (0.01-1 µM). However, 96 dose-dependently reduced UV-induced MMP-1 protein levels to normal levels by 73.1% at 0.01 µM [65]. In a dose-dependent manner, 96 effectively downregulated MMP-1 protein expression, whereas it upregulated type-1 procollagen protein expression in the UV-irradiated cultured human skin fibroblasts of an elderly person [66].

Matrix Metalloproteinase's Activity
Some triterpenoids (95-97, 100) were isolated and further tested the Matrix M proteinases (MMPs)' activity of the methylene chloride soluble fraction of a metha tract from the stems of S. japonica. Among them, 95 and 100 displayed effective cy activities against human dermal fibroblasts (IC50 = 20.0 and 1.12 µM, respectively) 9). In addition, 96 and 97 exhibited no cytotoxicity for the same cells at the test dose 1 µM). However, 96 dose-dependently reduced UV-induced MMP-1 protein levels mal levels by 73.1% at 0.01 µM [65]. In a dose-dependent manner, 96 effectively do ulated MMP-1 protein expression, whereas it upregulated type-1 procollagen pro pression in the UV-irradiated cultured human skin fibroblasts of an elderly person Styrax japonoside B (26) exerted inhibitory activity against MMP-1 and pre UV-induced changes in MMP-1 expression. At 10 µM, the treatment led to a sign dose-dependent reduction in MMP-1 protein expression, with an average decr 62.1% compared with the vehicle-treated control cells. The findings suggested t glycoprotein can potentially be used as a potent antimetastatic agent. This glyco exerts its effect by suppressing MMP-9 enzymatic activity through the NF-κB an signaling pathways [67].
Two cinnamyl esters (86 and 87) and seven pentacyclic triterpene acids (119 an 130) in Styrax are the key components that inhibit hCES1A activity. These Styrax japonoside B (26) exerted inhibitory activity against MMP-1 and prevented UV-induced changes in MMP-1 expression. At 10 µM, the treatment led to a significant dose-dependent reduction in MMP-1 protein expression, with an average decrease of 62.1% compared with the vehicle-treated control cells. The findings suggested that the glycoprotein can potentially be used as a potent antimetastatic agent. This glycoprotein exerts its effect by suppressing MMP-9 enzymatic activity through the NF-κB and AP-1 signaling pathways [67].

Antiasthmatic, Antiulcer, and Anti-Inflammatory Activities
In a murine asthma model, homoegonol (17) exerted significant effects in reducing inflammatory cell infiltration and Th2 cytokine production in the bronchoalveolar lavage fluid. It also attenuated airway hyperresponsiveness, decreased serum IgE levels, and downregulated iNOS and MMP-9 expression. Thus, compound 17 exhibited the potential to effectively suppress OVA challenge-induced asthmatic responses ( Figure 10). 2.2.11. Antiasthmatic, Antiulcer, and Anti-Inflammatory Activities In a murine asthma model, homoegonol (17) exerted significant effects in reducing inflammatory cell infiltration and Th2 cytokine production in the bronchoalveolar lavage fluid. It also attenuated airway hyperresponsiveness, decreased serum IgE levels, and downregulated iNOS and MMP-9 expression. Thus, compound 17 exhibited the potential to effectively suppress OVA challenge-induced asthmatic responses ( Figure 10).  In 2005, the extracted fractions of S. pohlii aerial parts, including the EtOAc fraction, ethanolic extract, and hexane fraction, were evaluated for their inhibitory activities against COX-1 and COX-2. The isolated products were further assessed against COX-1 and COX-2. The results revealed that all crude fractions and isolated products induced weak-tomoderate COX-1 and COX-2 inhibition. Among them, 57 exerted mild COX-1 inhibition, of 35.7% at 30 µM [69].

Other Activity
Through micronucleus and comet assays, Oliveira demonstrated that different doses (250, 500, and 1000 mg/kg body weight) of the S. camporum extract's compounds 4 and 17 had no genotoxic effect in Swiss mice. Moreover, they were effective in reducing doxorubicin-and methanesulfonate-induced DNA and chromosomal damage [11].
Braguine [69] investigated the EtOAc fractions of S. camporum and S. pohlii and isolated and identified compounds 152-155. Upon biological evaluation, they found that the EtOAc fractions, as well as compounds 152 and 155, could separate coupled Schistosoma mansoni adult worms. Additionally, compound 155 killed adult schistosomes in vitro. This research group also observed that homoegonol and homoegonol glucoside exhibited the best results against S. mansoni adult worms [70].
In vitro assessments were conducted to determine the protein tyrosine phosphatase 1B (PTP1B)'s inhibitory activities of compounds from S. japonica stem bark. Among the isolated compounds, 108 and 109 had the highest inhibitory activities (IC 50 = 7.8 and 9.3 µM, respectively) [45].
Jegosaponins A and B (158 and 159, respectively) exhibited potent hemolytic activity in sheep defibrillation (IC 50 = 2.1 and 20.2 µg/mL, respectively) and could improve the performance of PC-3 cells and zebrafish embryos through the identification of a membrane nonpermeable DRAQ7, which is a fluorescent nucleus staining dye [58] (Figure 11).

Materials and Methods
Through the search of a variety of online libraries such as Wiley Online Library, Pub-Med, Scifinder Web, ACS, and Web of Science, a summary of the newly discovered chemicals isolated from the genus styrax and their related biological activities in recent decades was provided. All species names were checked using http://www.theplantlist.org (accessed on 10 May 2023).

Conclusions
In summary, the Styrax genus comprises 130 species, and most of the species are

Materials and Methods
Through the search of a variety of online libraries such as Wiley Online Library, PubMed, Scifinder Web, ACS, and Web of Science, a summary of the newly discovered chemicals isolated from the genus styrax and their related biological activities in recent decades was provided. All species names were checked using http://www.theplantlist.org (accessed on 10 May 2023).

Conclusions
In summary, the Styrax genus comprises 130 species, and most of the species are extensively used as traditional medicines (Appendix A), particularly in China and Korea. Styrax can be easily collected because of its extensive distribution. All the species of the Styrax family, which were reported regarding the aspects of phytochemistry and pharmacology, were comprehensively summarized. In total, 159 compounds (Appendix B), including lignans, terpenoids, steroids, etc., were isolated from various species. The biological activities of those isolated compounds were subsequently investigated, exhibiting broad bioactivities such as cytotoxic activity, antioxidant activity, antifungal activity, apoptotic activity, anti-inflammation activity, anti-complement activity and so on. Chemical and pharmacological studies on the Styrax genus also proved that its main constituents are lignans and terpenoids. Moreover, several bioactive molecules exhibiting strong pharmacological activities were also isolated from Styrax (Appendix C).
Of note, information about the structure-activity relationships of most bioactive compounds is insufficient due to the lack of derivatives. Therefore, the exploitation of the versatility of the potentially bioactive natural compounds obtained from this genus is in great demand. Moreover, some species used in traditional medicines are still untapped such as S. suberifolius, which is used as a cure for rheumatic arthritis, whereas the modern physiochemical and pharmacological investigations are missing. Furthermore, in-depth pharmacological studies, especially in vivo studies, of the isolated biomolecules should be conducted in the future.           Jegosaponins B S. japonica [58] Appendix C Jegosaponins B S. japonica [58] Appendix C Jegosaponins B S. japonica [58] Appendix C           Jegosaponins B S. japonica [58] Appendix C